According to generally accepted knowledge, two classes of cell organelles, i.e. plastids and mitochondria, are derived from initially independent prokaryotes that were taken up into a predecessor of present day eukaryotic cells by separate endosymbiotic events (Gray, 1991). As a consequence, these organelles contain their own DNA, DNA transcripts in the form of messenger RNA, ribosomes, and at least some of the necessary tRNAs that are required for decoding of genetic information (Marechal-Drouard et al., 1991).
While, shortly after endosymbiotic uptake, these organelles were genetically autonomous since they contained all the elements necessary to drive prokaryotic life, this autonomy was reduced during evolution by transfer of genetic information to the cell's nucleus. Nevertheless, their genetic information is of sufficient complexity to make recent cell organelles an attractive target for gene technology. This is particularly the case with plastids, because these organelles still encode about 50% of the proteins required for their main function inside the plant cell, photosynthesis. Plastids also encode their ribosomal RNAs, the majority of their tRNAs and ribosomal proteins. In total, the number of genes in the plastome is in the range of 120 (Palmer, 1991). The vast majority of proteins that are found in plastids are, however, imported from the nuclear/cytosolic genetic compartment.
Plastids Can Be Genetically Transformed
With the development of general molecular cloning technologies, it became soon possible to genetically modify higher plants by transformation. The main emphasis in plant transformation was and still is on nuclear transformation, since the majority of genes, ca. 26,000 in the case of Arabidopsis thaliana, the complete sequence of which was recently published (The Arabidopsis Genome Initiative, 2000), is found in the cell's nucleus. Nuclear transformation was easier to achieve, since biological vectors such as Agrobacterium tumefaciens were available, which could be modified to efficiently enable nuclear transformation (Galvin, 1998). In addition, the nucleus is more directly accessible to foreign nucleic acids, while the organelles are surrounded by two envelope membranes that are, generally speaking, not permeable to macromolecules such as DNA.
A capability of transforming plastids is highly desirable since it could make use of the high gene dosage in these organelles that bears the potential of extremely high expression levels of transgenes. In addition, plastid transformation is attractive because plastid-encoded traits are not pollen transmissible; hence, potential risks of inadvertent transgene escape to wild relatives of transgenic plants are largely reduced. Other potential advantages of plastid transformation include the feasibility of simultaneous expression of multiple genes as a polycistronic unit and the elimination of positional effects and gene silencing that may result following nuclear transformation.
Methods that allow stable transformation of plastids could indeed be developed for higher plants. To date, two different methods are available, i.e. particle bombardment of tissues, in particular leaf tissues (Svab et al., 1990), and treatment of protoplasts with polyethylene glycol (PEG) in the presence of suitable transformation vectors (Koop et al., 1996). Both methods mediate the transfer of plasmid DNA across the two envelope membranes into the organelle's stroma.
Conventional methods for plastid transformation usually rely on the selection for the insertion of an antibiotic resistance marker cassette into the plastome such as an expression cassette containing the gene aadA (encoding the enzyme aminoglycoside adenyl transferase), which confers resistance to inhibitors like Spectinomycin or Streptomycin (U.S. Pat. No. 5,877,402) or aphA-6 (encoding the enzyme aminoglycoside phosphotransferase A-6) which confers resistance to kanamycin (Huang et. al., 2002). Alternatively, selection is achieved by replacing a complete resident plastid gene by a mutant gene which confers resistance to selection inhibitors (U.S. Pat. No. 5,451,513). These selection marker genes that are needed for the selection of transgenic plant cells from a vast background of untransformed cells code for antibiotic or herbicide resistance. The selection marker gene or the mutant plastid gene is included in the integrating region, which is flanked by homologous regions directing the plastome integration. Selection for plastid transformants is then achieved by cultivating transformed plant material on medium containing the appropriate inhibitor. As these marker genes are stably integrated into the genome together with the genes of interest, they will remain in the homoplastomic transgenic plants although they are not required for the function of the genes of interest. These remaining marker genes are a main issue of criticism of plant biotechnology. Construction of a selection system which does not result in a resistance gene in the transgenic plant is, therefore, highly desirable (lamtham and Day, 2000).
Conventional plastid transformation technology is described in Heifetz, 2000 and Koop et al., 1996.
Plastid transformation vectors usually contain one or more gene(s) of interest flanked by two regions of the insertion site, which are necessary for the stable introduction of the engineered sequences into the plastome by homologous recombination events (U.S. Pat. Nos. 5,877,402, 5,451,513). However, substantial cloning work is needed to generate the transformation vector molecules which contain a large number of different fragments: two flanks, a marker gene, one or more gene(s) of interest and regulatory elements such as promoter, 5′-UTR, 3′-UTR or spacer elements.
The cloning of transformation vectors is problematic in cases wherein (at least one of) the cloned gene(s) has a toxic effect on the bacteria used for cloning. Moreover, using the highly desirable potential to co-express a series of introduced transgenes is limited by the overall size of the transforming plasmid.
One major complication in achieving plastid transformation is the high copy number of the plastome. Following transfer of the vector DNA into the plastids only one or very few copies of the introduced molecules will recombine with the plastome. Thus initially only a small proportion of recombinant plastome molecules are generated in the background of a vast majority of wild type plastome molecules (“heteroplastomic status”). By a very time consuming process of segregation under selective pressure it is possible to eliminate the original wild type copies of the plastome and achieve a “homoplastomic recombinant status” being characterized by the sole presence of recombinant plastome molecules. Achieving the homoplastomic status is supported by several cycles of regeneration on selective medium containing the appropriate antibiotics. Usually 3-5 of such cycles are necessary to obtain the homoplastomic recombinant status. The presence of remaining copies of wild type plastome can be monitored by molecular analysis like PCR or Southern Hybridization. As several weeks are needed for one regeneration cycle it takes several months to generate hormoplastomic plastid transformants.
Therefore, it is an object of the invention to provide a novel, efficient, rapid and highly versatile process of genetic transformation of plant plastids, whereby genetically stable transgenic plants or plant cells may be produced.
It is another object of the invention to provide a process of genetic transformation of plant plastids, which allows a significant reduction of the number of regenerations cycles needed to achieve homoplastomic plants.
It is another object of the invention to provide a process of genetic transformation of plant plastids, which allows expression of multiple genes of interest (polycistronic expression).
It is a further object to provide vectors which can be used in a modular fashion, thus reducing the cloning work and the overall size of the plasmid molecules.
It is a further object to provide vectors which allow the cloning of sequences having toxic effects on the bacteria used for cloning.
It is a further object to provide a method that allows the generation of transformants which do not carry a resistance marker gene in the final plant or plant cell.